The habenular nuclei are part of an evolutionarily conserved conduction pathway from the limbic system to the midbrain. Although the habenulae have been implicated in diverse behaviors and in a variety of neurological disorders, a detailed understanding of the relationship between their organization and function is lacking. Zebrafish are a valuable system to study habenular structure and connectivity due to their amenability to transgenic approaches. Moreover, the zebrafish habenulae provide a unique model for studying left-right differences in the brain. The right and left habenulae consist of two anatomically distinct nuclei and show pronounced differences in size, neuropil density and gene expression, but how this asymmetry relates to subnuclear organization is also unclear. The overall goals of the proposed study are to characterize the organization and connectivity of the zebrafish habenulae in greater detail, and to analyze the functional relevance of habenular organization. Habenular subregions will be defined by the production of detailed gene expression and neuronal projection maps. New transgenic lines will be developed from the regulatory regions of genes expressed in distinct habenular nuclei or asymmetric subdomains to map projections of individual neurons using Brainbow and to selectively disrupt or inhibit a given habenular regions in behavioral studies. Behavioral assays will test the role of the dorsal habenular nucleus in anxiety and fear-avoidance assays. Understanding the functional significance of habenular organization will provide insight into how this clinically important brain region regulates behavior, as well as provide a unique model to understand the processing of lateralized information.